Enzymatic transition state structure can be experimentally approached by a combination of intrinsic kinetic isotope effects (KIEs) and computational quantum chemistry. Experimental KIEs provide boundaries for computational transition states. Transition state knowledge provides chemical insights and practical application of molecular electrostatic maps of transition states are blueprints for transition state analog design. Transition state analysis has provided femtomolar to picomolar inhibitors for N-ribosyltransferases, some of the most powerful enzyme inhibitors. Several transition state analogs are in human clinical trials with more in preclinical studies. These analogs follow the predictions of Pauling and Wolfenden that transition state mimics bind tightly by engaging the forces permitting enzymes to accelerate reactions up to 20 orders of magnitude. Yet, the application of transition state structure to inhibitor design is in its infancy, with development fr only a few chemical reaction classes. The fundamental processes leading to transition state formation remain in contention. This project will expand the utility of transition state analysis ad inhibitor design to two new reaction classes. A fundamental property of enzymatic transition state formation will be tested by individual heavy amino acid substitution into catalytic sites. Th first aim will target human phenylethanolamine N- methyltransferase (PNMT), the SAM-based formation of epinephrine. N-Methylation reactions are critical for hormones and neurotransmitter metabolism. PNMT is a target for blood pressure regulation and has potential links to Alzheimer's disorder. Hydrolysis of beta-lactamases is our second target class. New Delhi zinc metallo-beta-lactamase (NDM-1) and serine-beta- lactamase are clinically important targets for antibiotic resistance and provide new transition state insight for beta-lactam hydrolysis. The thir aim develops a new experimental approach to resolve fast (fs-ps) protein motions linked to transition state barrier-crossing. Pioneering enzyme chemistry experiments have revealed that increased protein mass slows on-enzyme chemistry. The ultimate extension of heavy-enzyme technology will replace selected amino acids specifically in the catalytic site with their heavy (2, 13C, 15N) counterparts by cell-free protein synthesis with a single amino acid substitution. This project advances transition state chemistry, transition state analog design and the fundamental properties of enzymatic catalysis.